This invention pertains to adenoviral capsids containing chimeric protein IX.
Based on the popularity of adenoviruses as gene transfer vectors, efforts have been made to increase the ability of adenoviruses to enter certain cells, e.g., those few cells it does not infect and those cells they do not enter as well as would be desirable, sometimes selectively, an approach generally referred to as xe2x80x9ctargeting.xe2x80x9d Such efforts largely have centered on biochemical alteration of the proteins of the adenoviral coat, which include the fiber, penton, and hexon, as well as proteins IIIa, VI, and IX. Of these proteins, the trimeric fiber is chiefly responsible for attachment and internalization of wild-type adenoviruses to their host cells. Within a mature adenoviral capsid, fibers are grounded by close attachment of one end to the penton, but the other end (xe2x80x9cknobxe2x80x9d) of the fiber does not contact any other capsid protein. Thus, the entire fiber forms a spike-like protrusion from the capsid, and amino acid residues within the terminal xe2x80x9cknobxe2x80x9d domain function as a ligand for cell surface proteins during the process of adenoviral infection. To a lesser extent, the penton also mediates viral attachment through interactions between RGD motifs of the penton and xcex1v-integrins present on virtually all cells.
Given its central role in wild-type adenoviral infection, the majority of the efforts aimed at targeting adenoviruses have focused on modification of the fiber protein (see, e.g., International Patent Applications WO 95/26412 (Curiel et al.), WO 94/10323 (Spooner et al.), WO 94/24299 (Cotten et al.), and U.S. Pat. No. 5,543,328 (McClelland et al.)). These efforts have proven disappointing, largely because they fail to preserve important fiber protein functions, such as stable trimerization and penton base binding (Spooner et al., supra). Moreover, replacement of the fiber knob with a cell-surface ligand (McClelland et al., supra) produces a virus only suitable for infecting a cell type having that ligand. Such a strategy produces a virus having many of the same targeting problems associated with wild-type adenoviruses (in which cellular tropism is mediated by a single protein), thus decreasing the flexibility of the vector. Moreover, due to the integral connection between the fiber trimerization and targeting functions, obtaining a functioning mutant fiber protein with substituted targeting is difficult. For example, removing the fiber knob and replacing it with a non-trimerizing ligand (e.g., Spooner et al., McClelland et al., supra) results in a virus lacking appreciable fiber protein. Considering these drawbacks, there exists a need for improved strategies for targeting adenoviral vectors.
The present invention provides a chimeric protein IX (pIX). The chimeric pIX protein has an adenoviral pIX domain and also a non-native amino acid. Where the non-native amino acid is a ligand that binds to a substrate present on the surface of cells, the chimeric pIX can be used to target vectors containing such proteins to desired cell types. Thus, the invention provides vector systems including such chimeric pIX proteins, as well as methods of infecting cells using such vector systems. These and other aspects of the present invention, as well as additional inventive features, will be apparent upon reading the following detailed description and reviewing the accompanying drawing and sequences.